Resource for marking clones on the fly 4th chromosome For over a century, studies employing Drosophila melanogaster have resulted in significant advances in our understanding of highly conserved cellular processes and signaling systems. In the area of human health, Drosophila genetics has been an effective means for identifying disease-associated genes and for providing insights into their mechanism of action. The fourth chromosome (IV) is the final frontier for genetic analysis in Drosophila. Small and devoid of recombination IV has been largely ignored. Nevertheless, the long arm of IV contains roughly 105 genes. 55% of these genes have obvious human orthologs and 67% of the human genes have a disease association. A complete understanding of multicellularity requires the genetic analysis of mutations in these genes. Somatic and germline clones are established tools for studying the functions of lethal mutations in flies. The MARCM system for tracking clones of mutant cells he has been widely employed to study central nervous system development and adult intestinal stem cells. However, MARCM is not useable on IV due to the lack of appropriate chromosomes. As a resource for the Drosophila community whose investigators are funded by virtually all of the NIH Institutes and Centers, we propose a collaborative R21 (in response to PAR-16-141) to generate the necessary chromosomes for MARCM-IV. Our two labs are currently NIH funded for studies of TGF-? signaling and have worked together previously on the role of the Sno oncogene in TGF-? signal transduction. We will employ an innovative strategy integrating molecular methods (Crisper-Cas9) with genetics (X to autosome jumping). MARCM-IV will then be tested in studies of TGF-? signaling in the mushroom body and the enteric neurons of the larval brain. The Specific Aims of this project are: Aim1 Resource Development: To create the unique fourth chromosomes necessary for MARCM-IV. Aim2 Discovery: Proof of principal studies will utilize MARCM-IV to generate marked clones for three mutant genes. These are the TGF-? ligands activin-? and myoglianin and the Smad-interacting signal transducer dCORL (fussel in Flybase). These applications of MARCM-IV will advance our knowledge of molecular mechanisms in the TGF-? pathway, neural-glial interactions and the development/function of subesophageal neurons in the brain. The results should attract the attention of others in the Drosophila community whose interests encompass genes on IV. We will provide the MARCM-IV lines to any qualified investigator and evidence of community interest is already visible in letters appended to this proposal. Given that many of the genes on IV are conserved, new insights from MARCM-IV in flies can be readily translated into new hypotheses for normal development/physiology or diseases in humans. Looking ahead, it is likely our innovative approach can easily be expanded to an analysis of all genes on IV. The valuable community resource created by this project will be made freely available to all qualified researchers to facilitate our understanding of conserved features of developmental signaling and neurobiology impacting human health and disease.